Abstract The inflammasomes are crucial innate immune signaling platforms implicated in immune defense against infections and autoimmune/autoinflammatory disorders such as multiple sclerosis, diabetes, and Alzheimer?s disease.Pyroptosisistriggeredbyinflammatorycaspases-1,4,5,or11uponstimulationofthecanonicaland noncanonicalinflammasomes.DespitetherecentprogressontheidentificationandcharacterizationofGSDMD asaneffectorofpyroptosis,themoleculareventsunderlyingthetransformationofGSDMDfromanautoinhibited restingstatetotherecognitionandcleavagebycaspaseshasremainedpoorlydefined.Thelackofmechanistic understanding of GSDMD-mediated pyroptosis has hampered progress on the development of novel therapeuticsagainstinfectionsandautoimmune/autoinflammatorydisorders.Thisproposaltargetscriticalgaps in our understanding of GSDMD/inflammatory caspase-mediated pyroptosis using complementary structure- functionapproaches.WehypothesizethatGSDMDismaintainedinanautoinhibitedconformationthrough intramoleculardomaininteractionsbetweenitsN-(GSDMD-N)andC-terminal(GSDMD-C)domains.Such conformation is recognized by inflammatory caspases that cleave at the linker region to release the autoinhibition and facilitate membrane pore formation. We propose the following specific aims to test the abovehypothesis.Aim1.DefinethemechanismthroughwhichGSDMDmaintainsitsautoinhibitedstate,and howsuchautoinhibitionmayberegulatedbysmallmoleculetoolcompounds.Wewillcharacterizethestructure oftheGSDMDdomainsandthefull-lengthproteininitsautoinhibitedstateusingprimarilystructuralapproaches, inconcertwithanalysisoftheGSDMD-NandGSDMD-Cinteractionsthroughbiochemicaltechniques.Therole of the intramolecular domain interface during Salmonella infection-induced pyroptosis will be probed using GSDMD-deficient cell lines reconstituted with mutant GSDMD. A unique feature of our approach is the incorporationofGSDMD-bindingcompoundsidentifiedfromchemicallibrariesscreeningintotheaboveefforts to investigate the regulation of GSDMD and pyroptosis by small molecule tool compounds. Aim 2. Define the mechanismsofgasderminrecognitionandcleavagebyinflammatorycaspases.Wewillelucidatethemolecular basis of the conversion of caspases-4 and 11 from zymogens to active conformation, which has been poorly characterized.WewillinvestigatethemechanismofGSDMDrecognitionbythesecaspases,whichmayinvolve boththelinkerpeptidesandtertiarystructureofGSDMD.Insummary,thisprojectwilldelineatethemolecular mechanisms underlying the autoinhibition and activation of GSDMD during pyroptosis, identify small molecule toolcompoundsandcharacterizetheirmodeofactioninthecontextofpyroptosisregulation.Thiswillnotonly furnish unique insights on GSDMD structure and function, but may stimulate the development of therapeutics thattargetinflammatorydisorders.